California Avocado Society 1976 Yearbook 60: 160-163
RESPONSES OF AVOCADO STEM PIECES IN TISSUE CULTURE
C. A. Schroeder
Department of Biology, University of California, Los Angeles
The techniques developed for the establishment of avocado clones by grafting, budding
and by the rooting of leafy stem cuttings are well described in the literature (2, 4) and
are presently widely utilized by nurserymen throughout the world. Standard nursery
practices involve the use of germinated seed for rootstock source onto which are
budded the selected clonal materials. Rooted cuttings can be obtained directly from
some old clones by use of leafy cuttings assisted by root promoting hormones. Other
old avocado clones usually are more difficult to root, as cuttings can be established on
their own roots by the special techniques utilizing a treatment of etiolation or growing
the clonal tissue in the dark for at least a portion of time before the roots are induced to
form (2).
The objective of developing techniques to obtain clonal material of avocado by apical
meristem culture for rootstock or other purposes has been the focus of attention by
many investigators. Attempts to approach this problem by the use of tissue culture
techniques have been encouraged by the success attained with other plant materials,
usually those more succulent or herbaceous in nature, such as chrysanthemums,
pelargonium, carnations, etc. (1, 5, 6). Fast growing species are comparatively easy to
propagate by conventional methods, namely the rooting of leafy cuttings, and thus have
proved feasible to propagate by culture of the apical meristem, which, if accomplished
under appropriate conditions results in virus-free plants. Woody plant species on the
other hand are usually slower in their growth responses and, likewise have proved more
difficult to establish as clonal material from apical meristem or from small apical tips (3).
The investigations reported here are presented as a progress report on some studies
which have been conducted on avocado tissues grown in vitro. Previous reports (7, 8, 9,
10) have been made which indicated that tissues from nearly all parts of the avocado
plant can be cultured as callus masses in vitro. These materials have been studied from
several points of view and with different objectives in mind. Some of the results attained
thus far are only encouraging and do not necessarily point to definite conclusions but do
suggest that useful techniques might be developed which may be helpful in propagation
procedures. It is quite possible to maintain cultures of callus tissue derived from
avocado the cotyledon for periods of fifteen years or more according to observations
made at present. It is conceivable that callus derived from other tissues likewise, might
be maintained indefinitely. Such tissue does not exhibit obvious conditions of
differentiation but is merely continued in culture as a callus mass by a series of divisions
into smaller segments and transfer of these segments to fresh media at various
intervals of time, usually one to three months. Differentiation of this apparently
homogeneous callus mass into definite, recognizable structures such as roots has been
observed in many experiments. The development of shoot structures, per se, from
callus tissues has not yet been noted under the conditions of the present investigations.
While the reproduction of an entire and normal plant from callus tissue derived from a
relatively pure specific tissue of avocado has not yet been achieved, such an objective
has been attained in several species such as tobacco (4), carnation (6) and others (1). It
appears to be theoretically possible to accomplish the re-constitution of an avocado
plant from any of its cells or a small group of cells, but the practical problems have not
been overcome at this time. Therefore attempts are under way to reproduce avocado
plants from more organized tissues, such as, apical meristem or small buds.
Repeated attempts have been made to culture in vitro what are considered as normal
apical or lateral buds obtained directly from trees in the field. These buds did not
indicate any evidence of length growth when collected but were "plump" and otherwise
in a healthy condition. The buds at this stage are about three to four millimeters in
length. They consist of many small bud scales and a few larger protective bud scales
which cover the delicate apical meristem and sub-apical tissues. The selection of tissue
in this condition is not considered as a meristem culture technique. The materials have
been selected at this stage in an attempt to establish in vitro the more gross structure of
an intact bud. True apical meristem tissues less than one millimeter in length have been
excised from buds under the microscope, but, have failed to survive under a limited
number of conditions which have been tried. This aspect of the study will require more
refinement of techniques and must be repeated under a wider range of conditions.
The general behavior of material taken as "intact buds" when planted on a wide range
of nutrient agar media, has been noted to proliferate as a callus from the cut surface or
more usually from the periphery of the cut surface, when the bud is placed with the cut
surface on the agar media. Massive callus formation, the result of extensive cell
division, will generally cover the agar surface within the culture vial. This is followed by a
considerable increase in the thickness of the callus mass which grows upward,
generally enveloping and hence "inundating" the original bud mass, such that it cannot
be detected within the callus. Occasionally some of the buds will exhibit some length
growth and will eventually protrude from the callus mass to a distance of four to five
millimeters. This length growth appears to be limited or inhibited in some manner,
hence, the culture makes little progress except to increase the size of the callus.
Another approach to the problem of establishment of lateral or apical buds in vitro,
which has provided more response in the development of length growth, has been the
pretreatment of the bud system before excision of the bud. The general practice has
been to place small grafted trees or seedlings in half gallon or gallon cans in a dark
walk-in room. The leaves are removed from the stems and the new lateral shoots are
allowed to grow in the dark as etiolated tissues. Considerable length growth of a lateral
bud up to half a meter (18 inches) or more gives rise to a thin white lateral shoot with
many small white leaves, each usually less than one centimeter in length. This etiolated
stem will have a bud in the axil if each leaf, such that 20 to 25 lateral buds may be
obtained from a half meter length of stem. The stem is generally cut into small cylinders,
five to ten millimeters in length. Each cylinder may or may not contain a lateral bud and
subtending leaf.
These etiolated cylinders of tissue are placed on their side on the agar nutrient media of
several compositions and under various conditions of temperature and light. The first
response of the tissue is that of massive proliferation at the cut ends to produce a
callus. There is, however, a stronger tendency for the lateral bud, when present, to
elongate and produce measurable length growth. The extension of the bud results in the
normal development of small leaves, hence, a normal but small plantlet is established,
but with a massive callus at the base. Occasional roots will eventually develop in some
of the cultures in which length growth of the apical portion has been achieved. Root
differentiation and development has not been induced at will under the present
experimental conditions.
LITERATURE CITED
1. BEN-YA’ACOV, J. and R. W. LANGHANS. 1972. Rapid multiplication of
chrysanthemum plants by stem-tip proliferation. Hort. Sci. 7: 289-90.
2. FROLICH, E. F. and R. G. PLATT. 1972. Use of the etiolation technique in rooting
avocado cuttings. Calif. Avocado Soc. Yearbook 55: 97-109.
3. GRINBLAT, U. H. 1972. Differentiation of Citrus stem in vitro. Jour. Amer. Soc. Hort.
Sci. 97: 599-603.
4. HARTMANN, H. T. and D. E. KESTER. 1975. Plant Propagation Principles and
Practices (3rd Ed.) Prentice-Hall, Englewood Cliffs, N.J.
5. Hollings, M. 1965. Disease control through virus-free stock. Ann. Rev. Phytopath. 3:
367-396.
6. MURASHIGE, T. 1974. Plant propagation through tissue cultures. Ann. Rev. Plant
Physiol. 25: 135-166. 1974.
7. SCHROEDER, C. A. 1968. The longevity of avocado tissue in vitro. Calif. Avocado
Soc. Yearbook 52: 123-130.
8. SCHROEDER, C. A. 1971. The response of avocado pericarp tissue to temperature
and light in vitro. Calif. Avocado Soc. Yearbook 54: 85-89.
9. SCHROEDER, C. A. 1973. The response of apical meristem and other tissues of
avocado in aseptic culture. Calif. Avocado Soc. Yearbook 56: 138-141.
10. SCHROEDER, C. A. 1975. Response of avocado flower buds and floral parts
cultured in vitro. Calif. Avocado Soc. Yearbook 58: 66-73.